Process for simultaneously removing leukocytes and methylene blue from plasma

ABSTRACT

Filter devices for simultaneously removing leukocytes and viral inactivating agents from whole blood or blood fractions are disclosed. One type of device comprises (1) a housing surrounding (2) activated carbon and (3) a mechanically stable polymeric material which may optionally be modified to attach a ligand for leukocytes. General methods for removing leukocytes and viral inactivating agents from blood and plasma are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of our U.S. application Ser. No. 08/347,564,filed Nov. 30, 1994, now U.S. Pat. No. 5,639,376, which is acontinuation-in-part of our U.S. application Ser. No. 08/204,102, filedMar. 1, 1994, abandoned, which is a continuation-in-part of our earlierapplication Ser. No. 08/179,567, filed Jan. 10, 1994, abandoned, theentire disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to filters for removing leukocytes and viralinactivating agents from whole blood or blood fractions, and methods forusing the filters to remove leukocytes and viral-inactivating agentsfrom whole blood or blood fractions.

BACKGROUND OF THE INVENTION

A significant risk associated with the use of blood products intransfusion medicine is the presence of viruses in such products aswhole blood, plasma, platelets, and various blood fractions. A number ofchemicals have been identified as viral inactivating agents; theiraddition to the blood product, sometimes in conjunction with irradiationby visible and/or ultraviolet light, causes viruses to lose theirinfectivity. For example, methylene blue is added to plasma intended fortransfusion. Although methylene blue exhibits effective virucidalactivity and is considered generally safe, it nevertheless represents anexogenous component in the plasma with possible long-term adverseeffects not yet fully understood. Other viral inactivation agents suchas psoralens carry similar risks. One object of the present invention isto offer a method for removing antiviral agents after their virucidalfunction is completed.

European application 239,859 describes a method that is currentlyemployed to remove lipid soluble process chemicals from biologicalfluids. It comprises bringing the fluid into contact with a naturallyoccurring oil, agitating the resultant mixture, separating the phases bysedimentation or centrifugation, decanting the upper lipid phase, andutilizing the residual biological fluid. Aside from the mechanicalcomplexity of this process, it appears applicable only to the removal oflipid soluble process chemicals.

Gel filtration is also known for removing small molecules from bloodfractions based on molecular weight differences. Horowitz et al.Transfusion, 25, p. 516-522 (1985)! have described the removal oftri-n-butyl phosphate from anti-hemophilic factor concentrates bychromatography on Sephadex G-25; however, gel chromatography is not apractical method for removing small molecules from plasma and wholeblood.

PCT application WO 91/03933 discusses the use of silica gel, modifiedsilica gel, glass beads, and amberlite resins to adsorb methylene bluefrom plasma. None of the methods presently in use or proposed isparticularly attractive for the routine processing of plasma.

A second concern that arises with blood products, including those suchas plasma, is their non-homogeneity; blood products commonly includeseveral cell types as well as a variety of molecular components havingdiffering biological activities. Often patients into whom the bloodproduct is to be transfused are only in need of one component, and theother components present in the blood product are not only unnecessarybut may even be disadvantageous or harmful. In this respect, leukocyteshave come to be regarded as unwanted passengers in transfusions because"once transfused, they may turn upon their host and-unleash endogenouspyrogens, cell-associated viruses, or even lethal graft-versus-hostdisease." see Klein "Wolf in Wolf's Clothing: Is It Time to Raise theBounty on the Passenger Leukocyte?" Blood 80, 1865-1867 (1992)!. Forthis reason it is desirable that leukocytes be reduced to the lowestfeasible levels. It would therefore be highly desirable to have a methodfor removing leukocytes from plasma quickly and efficiently. Moreover,there is a need for a simple and effective method for simultaneouslyremoving viral inactivating agents and leukocytes from plasma.

Media and devices for removing leukocytes from red blood cellconcentrates, platelet concentrates, and other blood fractions have beendescribed. The media are typically non-woven mats of controlled fiberdiameter. They are adequate as components for fabricating a deviceaccording to the invention, but media for filtration based primarily onseparation by size can be improved by adding ligands for leukocytes, asdescribed in our earlier copending application Ser. No. 08/179,567.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a filter forremoving both leukocytes and viral inactivating agents (such asmethylene blue and its metabolites and photodecomposition products) fromplasma or other blood fractions quickly, safely and with very highefficiency.

It is a further object to provide a method of removing leukocytes andviral inactivating agents from whole blood, plasma or other bloodfractions. It is an advantage of the invention that the foregoingobjects can be accomplished quickly, thoroughly and inexpensively.

The present invention permits efficient, integrated (i.e., single-step)removal of both leukocytes and virus-inactivating compounds fromplasma--thus producing a blood product free of active viruses, antiviralcompounds, and leukocytes.

These and other objects, features and advantages are realized by thepresent invention which relates to a method for simultaneously removingleukocytes and one or more viral inactivating agents from blood.products, such as plasma. The method comprises passing the plasmathrough a filter adapted for removing leukocytes and antiviral agents."Adapted for removing leukocytes" means having appropriate geometry andsurface chemistry to trap at least a portion of available leukocyteswhile allowing other blood components of interest to pass. Removal ofantiviral agents is achieved by means of sorption onto activated carbonor media containing activated carbon.

In one aspect the invention relates to a method for simultaneouslyremoving leukocytes and one or more viral inactivating agents from wholeblood or a blood fraction comprising passing the blood or blood fractionthrough a filter adapted for removing leukocytes and antiviral agents,said filter comprising (1) a mechanically stable polymeric materialcapable of retaining leukocytes and (2) activated carbon capable ofremoving the viral inactivating agent. A portion of the mechanicallystable polymeric material may optionally have covalently attachedthereto a first ligand, which has affinity for a leukocyte cell surface.In a preferred method, the blood fraction is plasma. In anotherpreferred method pertaining specifically to plasma, the viralinactivating agent is selected from the group consisting ofphenothiazine dyes and photodecomposition products of phenothiazinedyes. Preferably, the viral inactivating agent is selected from thegroup consisting of methylene blue, toluidine blue, andphoto-decomposition products of methylene blue and toluidine blue. Themechanically stable polymeric material that retains leukocytes may beincluded within a laid textile web.

In a specific embodiment, the blood or blood fraction is passedsequentially through (1) a layer containing activated carbon and (2) atleast one shape-sustaining laid textile web having a thickness of 1 to 8mm and a bulk density of 0.05 to 0.4 g/cm³. The web is made up of:

(a) a plurality of interlocked textile fibers with average deniersbetween 0.05 and 0.75 and average lengths between 3 mm and 15 mm. Thetextile fibers are substantially uniformly distributed in the web so asto form a matrix of the textile fibers with spaces between adjacentinterstices of interlocked fibers; and

(b) a mechanically stable polymeric material comprising a plurality offibrillated particles of polymeric material having a surface area of 5to 60 square meters per gram substantially disposed within the spaces ofthe matrix. The fibrillated particles have a plurality of fine fibrilswhich are interlocked with adjacent textile fibers of the spaces suchthat the fibrillated particles are not substantially displaceable fromthe web during filtration.

In another aspect the invention relates to a filter device for removingleukocytes and one or more viral inactivating agents from whole blood ora blood fraction, comprising (1) a housing, enclosing (2) an activatedcarbon-containing filter element and (3) at least one filter elementadapted for retaining leukocytes. The filter element for retainingleukocytes may comprise a laid textile web which may optionally includea mechanically stable polymeric material having attached thereto a firstligand, which has affinity for the leukocyte cell surface. The firstligand may be attached directly to the polymeric material, or it may beattached to the polymeric material through at least one interveninglinker. When present, a preferred ligand is heparin.

In a specific embodiment, the filter device comprises (1) an activatedcarbon-containing filter element, preferably a carbon/cellulosecomposite, and (2) a shape-sustaining laid textile web as describedabove wherein the weight ratio of the fibrillated particles to thetextile fibers is between 1:99 and 40:60. A preferred polymeric materialis cellulose acetate and preferred textile fibers are polyolefin andpolyester fibers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic cross-sectional view of a filter deviceaccording to the invention.

FIG. 2 is a schematic representation of a filter medium according to thedisclosure of parent application Ser. No. 08/179,567 showing attachedligands for leukocyte surface features.

DETAILED DESCRIPTION INCLUSIVE OF PREFERRED EMBODIMENTS

In recent years there has been great interest in inactivating virusessuch as Hepatitis B (HBV), Hepatitis C (HCV), Human T LymphotrophicRetrovirus Type 3 (HTLV), Human Immunodeficiency Virus (HIV), andLymphadenopathy Associated Virus (LAV) in blood and blood products. Atpresent, methods for inactivating these viruses in blood and bloodfractions include treatment with a chemical disinfectant such asformaldehyde (see U.S. Pat. No. 4,833,165); treatment with solvents anddetergents (see U.S. Pat. No. 4,540,573) and treatment withphotosensitizers. For example, U.S. Pat. No. 5,232,844 describes the useof phthalocyanines; U.S. Pat. No. 5,041,078 describes the use ofsapphyrins; U.S. Pat. Nos. 4,169,204, 4,294,822, 4,328,239 and 4,727,027describe the use of various furocoumarins (psoralens) and analogsthereof; Meruelo et al. Proc. Nat. Acad. Sci. U.S., 85, 5230-5234(1988)! have described the use of hypericin; Lambrecht et al. Vox Sang.60, 207-213 (1991)! and Mohr and Lambrecht PCT application WO 91/03933!have described the use of phenothiazine dyes (methylene blue andtoluidine blue); and U.S. Pat. No. 4,915,683 describes the use ofmerocyanine dyes to inactivate viruses. According to these methods, anexogenous photosensitizer is added to the blood or plasma and thesolution is irradiated with light of appropriate wavelengths toinactivate the virus.

Phenothiazine dyes are photochemicals that bind to nucleic acids. Undersuitable activation conditions such as long-wavelength UV irradiation,phenothiazine dyes crosslink the DNA and RNA strands in viruses, therebydisabling uncoiling and replication. They also react with membranestructures and they induce the production of virucidal oxygen radicalsfrom molecular oxygen. These characteristics of phenothiazine dyes formthe basis of viral inactivation and certain photochemotherapies. See PCTapplication WO 91/03933.! However, the slight excess of phenothiazinedyes used to ensure thorough interaction with viruses and the consequentresidue left in the plasma represents some risk to the patient upontransfusion. For example, methylene blue has been suggested to possess acertain level of mutagenicity and other adverse effects may become ofconcern with long-term exposure associated with regular transfusion. Itis therefore desirable to remove the unreacted phenothiazine dyes ortheir metabolites and photodegradation products from the plasma afterthe viral inactivation treatment.

In all of the antiviral treatments, exogenous agents are added to thebiological fluid. In most cases, these exogenous agents must be removedfrom the biological fluid before it can be administered to a human. Thepresent invention entails the perfusion of the biological fluid throughan appropriately sized filter, which captures both leukocytes and viralinactivating agents. In some embodiments the filter may be designed toenhance the removal of leukocytes through the use of a matrix which issurface treated with carbohydrate-based ligands.

In the case of phenothiazine dyes used as the viral inactivating agents,single donor units of plasma are individually injected with preciselymeasured amounts of the dye, and mixed thoroughly inside the blood bag.The entire blood bag is then irradiated with fluorescent light ornarrow-band red light from light emitting diodes for a prescribed periodof time. This practice is fundamentally different from the batchwisetreatment of pooled plasma, i.e. large volumes of plasma obtained bycombining many single-donor units. Pooling is convenient from aprocessing scale viewpoint, but has the disadvantage that asingle-infected unit of plasma, ie. one carrying pathogens, is capableof contaminating an entire plasma pool. Single-donor unit processingavoids this risk; the practice is also particularly suited forsubsequent viral inactivating agent removal with an individual,disposable filtration device to result in a higher quality, individuallyidentifiable unit of plasma.

A filtration device may be sized according to the quantity of treatedplasma requiring methylene blue removal. Preferably, the device isdesigned to remove essentially all of the viral inactivation agent usedto treat a single unit of plasma--a highly desirable practice renderedfeasible by the methylene blue technique, for example.

In this invention, plasma that has been viral inactivated with methyleneblue is brought into contact with a filter medium containing activatedcarbon in a flow-through device. The activated carbon may be in the formof a discrete sorption layer of powder, granules, fibers, or fabric(woven, knitted, or nonwoven). Alternatively, carbon fibers (filamentsor staples) may be incorporated into a filter matrix as one of itscomponents. Another medium may be a porous solid comprising activatedcarbon as its active ingredient. Yet another medium may be a compositestructure, combining one or more forms of activated carbon with othernon-carbonaceous structural elements, to provide filtration media withspecific sorption, permeability and mechanical properties. In all cases,sorption of methylene blue will take place primarily on the activatedcarbon surfaces.

In the flow path of the filter device, the activated carbon medium maybe preceded by a depth filter with the capability of removing lipids andsolid impurities which may be present. Alternatively, the activatedcarbon medium itself may be constructed so as to impart lipid and solidretention properties. Another filter may optionally be placed downstreamof the activated carbon media to retain fragments or particles that maybe released from any of the filter components.

A hydrophilic coating may optionally be applied to the activated carbonsurfaces. This coating serves one or more of the following functions: 1)to encapsulate and contain the carbon material, thus reducing release offine particulates into the filtered plasma; 2) to reduce undesirableinteraction between the activated carbon and plasma components byoffering a biocompatible surface in direct contact with the plasma;and/or 3) to reduce or prevent sorption of species substantially largerthan methylene blue by means of size exclusion, viz. allowing relativelyunimpeded permeation of methylene blue and photolytic products comparedto that of larger molecules. The "cutoff" molecular weight of thespecies to be excluded may be controlled by varying the composition ofthe encapsulating layer. This is a method of reducing binding ofdesirable proteinaceous components in the plasma, such as coagulationfactors, by the activated carbon. Similar considerations apply to theremoval of viral inactivation agents other than phenothiazine dyes.

Plasma samples, especially those collected as single-donor units,exhibit a range of properties, the most readily noticeable of which isthe presence of chylomicrons. Chylomicrons include a range of lipidspecies of different sizes and degree of agglomeration. Units of plasmaheavily laden with chylomicrons become more noticeably tinted bymethylene blue because of preferential sorption of the dye by the lipid,and are correspondingly more objectionable to the user. In addition,such units are more challenging to filter because they tend to clog thepores of filter media.

To reduce the effect of clogging and ensure filtration of a single-donorunit of plasma can be completed within a reasonable time requires thatsufficient frontal area be available in the filter. This influences thedesign of the filter device in terms of packaging the necessary quantityof sorption media into the most favorable aspect ratio, i.e. the ratioof frontal surface area to volume. The filter media may be shaped aslayers of flat sheets, or as hollow fibers or cylinders where the plasmaflow would be directed through their annular walls.

Activated carbon media offer the advantage of a high-capacity sorbent,which translates to compact filter devices with small holdup volumes,and thus high recovery of the plasma product. With specific grades ofactivated carbon and/or by applying surface coatings, selective sorptionproperties may be created to allow removal and retention of differenttarget components in the treated plasma.

To minimize risks associated with repeated transfusion in long-termtherapy, it is prudent to remove as much methylene blue and itsphotoreaction byproducts (e.g. Azure B, Azure C) as possible after theviral inactivation step, preferably to levels below detectability (ca.0.02 μg methylene blue/mL plasma). An example of a suitable medium forthis purpose is a carbon composite medium in which activated carbonparticles are uniformly dispersed and embedded in a cellulose fibrousmatrix.

Much of the methylene blue added to plasma becomes associated with thechylomicron or endogenous lipids. Effective clearance of the dye fromthe plasma therefore also requires simultaneous removal of this lipidfraction. In addition, effective lipid removal would enable excessivelylipemic plasma units previously rejected to be processed fortransfusion. A fibrous, porous matrix made of lipophilic (i.e.hydrophobic) materials is appropriate for removing plasma lipids. Sinceremoval is accomplished by adsorption and size exclusion mechanisms,preferred media include those with relatively high surfacearea-to-volume ratios, morphologies favorable to depth filtration (e.g.decreasing effective pore diameter in the direction of flow through thethickness of the filter), and good biocompatibility to prevent excessivenon-specific protein adsorption.

Various coagulation factors may be depleted by non-specific adsorptionon the filter media. The consequences vary. For example, loss of FactorVIII is less significant than a comparable loss of Factor V, because theformer may be replenished using commercially available plasma fractionpreparations, while the latter is not. An ideal filter device shouldminimize changes in coagulation factor content of the plasma before andafter filtration. In practice, some modest degree of removal ofcoagulation factors may be tolerated because an excess of such factorsis present in the human body, and because the volume of plasmatransfused typically represents a small fraction of the total plasmavolume in the circulatory system.

Platelet-poor plasma used for transfusion typically has a leukocyteburden of about 10⁶ per mL. A 3-log reduction to 10³ per mL is generallyconsidered adequate for transfusion purposes. With the device and methodof the invention, leukodepletion may be performed simultaneously withmethylene blue removal after methylene blue treatment. Leukodepletionafter methylene blue treatment allows both the plasma-borne andleukocyteborne viruses to be inactivated simultaneously. Studies haveshown that a methylene blue concentration of 0.1 μM is adequate for bothpurposes.

There is some evidence that platelets may be activated by contact withcarbon particles. This problem may be addressed effectively in two ways:by coating the carbon surface with a more biocompatible material asdiscussed above, or by removing the platelets altogether from the plasmaby sorption onto appropriate depth filter media.

The filter comprises (1) a mechanically stable polymeric material, whichmay have a surface chemistry adapted for removing leukocytes, and (2)activated carbon or a medium containing activated carbon for removingviral inactivating agents. In one embodiment, at least a portion of themechanically stable polymeric material has covalently attached a firstligand having affinity for the leukocyte cell surface.

A suitable leukocyte depleting medium for use in the device of theinvention may comprise a laid textile web which includes a mechanicallystable polymeric material. In an improved medium, a portion of thepolymeric material may optionally have covalently attached thereto afirst ligand, which has affinity for a leukocyte cell surface. In oneembodiment, the first ligand may be attached directly to the polymericmaterial; in another, the first ligand is attached to the polymericmaterial through at least one intervening linker.

The first ligand may be a glycoprotein of the selectin family, or acarbohydrate, particularly a sulfoglycan that includes residues ofglucuronic acid, such as a heparin. The intervening linker may be theresidue of an alkylene diamine, in which case, the linker may beattached to the ligand by an amide bond to a carboxyl of the ligand,when one is present.

In the case of heparin, chondroitin sulfate and similar glycans bearingcarboxylic acid residues, the carboxylic acid may be activated forreaction with a nucleophile in the linker or polymeric material. Usuallythe nucleophilic residue is a primary amine and the activation utilizesany of the procedures well known in the art for forming amide bonds. Wehave found that EEDQ and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC) are particularly useful.

Other ligands now known or subsequently discovered are expected tofunction similarly. The critical requirements of the leukocyte ligandare a high affinity for the leukocyte surface and a functionalizablesubstituent at some position remote from the binding region whereby theligand can be covalently bound to the polymeric material.

In operation, an individual unit of plasma (or if necessary, some largervolume of plasma being processed) that is suspected of beingcontaminated by a virus will be treated by the addition of an effectiveconcentration of a virus-inactivating phenothiazine dye to the plasma.The unit of plasma is then irradiated for a sufficient time to permitthe antiviral compound to inactivate both "free" (i.e., plasma-borne)and cell-associated virus.

Next, and involving the methods and devices of the present invention,the treated plasma, still containing at least significant amounts ofantiviral agent, will be passed through the leukocyte/antiviral filterof the present invention, to produce a plasma product substantially freeof active viruses, leukocytes capable of harboring them, and residualsof the antiviral compound itself.

Filters that can remove leukocytes are known in the art. For example,Lydall Inc. manufactures a suitable leuko-depletion filter, and AsahiChemical manufactures another. More efficient filters activated byattachment of heparin and other ligands capable of enhancing the captureof leukocytes are disclosed in our earlier, copending U.S. applicationSer. No. 08/179,567.

One filter element useful in the fabrication of the filter of thepresent invention is a modification of the filter described in U.S. Pat.No. 5,190,657, the disclosure of which is incorporated herein byreference. Briefly, the filter consists of a filter material which is ashape-sustaining laid textile web. The web will commonly be cut in acircular configuration to form the filter and is suitable for loadinginto a cylindrical filter carrier.

The thickness of the web is at least 1 millimeter, most preferably atleast 2 millimeters, and can be up to about 8 mm. The density of thelaid web is between about 0.05 and 0.4 g/cm³.

The filter material is comprised of a plurality of matrix textilefibers, and these textile fibers have average deniers between about 0.05and 0.75. At least 60%, preferably at least 70% and more preferably atleast 80 to 85% of the fibers have deniers within the above-notedranges, and lengths from 12,000 to 180,000 m/g. The textile fibers aresubstantially uniformly distributed through the web so as to form amatrix of the textile fibers. The matrix has spaces between adjacentinterstices of the interlocked fibers. Within these spaces, there are aplurality of fibrillated particles of very high surface area. Thefibrillated particles are disposed within spaces, as well as along andamong the matrix textile fibers.

The matrix textile fibers are commonly synthetic polymer fibers, such aspolyolefin or polyolefin-sheathed fibers, polyamide, polysulfone,polyester, polyvinyl alcohol and poly(ethylene-vinyl alcohol) copolymerfibers. Polyolefin fibers are preferred.

The fibrillated particles are polyester fiber material, acrylic fibermaterial, nylon fiber material, polyolefin fiber material or cellulosicfiber material. Cellulose acetate is usually used since a great numberof fibrils are produced with that material, and the material has anatural hydrophilic nature.

FIG. 2 illustrates, in a highly idealized schematic form, one embodimentof a filter element useful in the filter of the present invention. Thefilter is comprised of a polymeric material 14 to which are covalentlybonded a plurality of ligands 15 for leukocyte cell surface features. Inuse, the leukocytes 11 are held within the filter matrix both bymechanical effects (size) and by specific interactions between bindingsites on the ligands 15 and sites on the surface of the leukocytes.

The filter elements used in the examples that follow are commerciallyavailable from Lydall Inc. Hamptonville, N.C.! and consist ofpolypropylene fibers and fibrillated cellulose acetate "fibrets." Ingeneral, any filter comprised of a cellulose acetate component and ashape-sustaining web that is resistant to base hydrolysis will functionin the methods described in application Ser. No. 08/179,567 forpreparing surface-modified filters, or may be used directly as aleukodepletion medium without further modification.

The particular filter device described below as a preferred embodimenthas the additional feature that it also removes chylomicrons,microaggregates, bacteria and endotoxins from plasma. The combinedeffects of the various features of the device are quite profound: (a) Byremoving >95% of methylene blue and its photolysis products, iteliminates concern about methylene blue toxicity and concern about thevisual appearance of the plasma; (b) by removing >99.9% of leukocytes,it improves virus inactivation capability, reduces leukocyte-associatedbacteria (e.g. Yersinia histolytica), and reduces leukocyte-associatedimmunologic effects; (c) by removing chylomicrons or lipids it improvesthe appearance of the plasma, eliminates the need for a microaggregatefilter at the bedside, and avoids having to discard highly lipemicplasma units; (d) by removing bacteria, it reduces sepsis; and (e) byremoving endotoxins, it reduces or eliminates febrile. reactions. All ofthese advantages are accomplished at low cost, with a plasma volume lossof less than 5%, and the process can be carried out on a single donorunit basis, thereby avoiding the hazards associated with pooling bloodsupplies.

EXAMPLES

A series of devices have been developed which incorporate multiplefunctions described above. These were evaluated for several keyperformance criteria; extent of methylene blue removal, time forfiltering a unit of plasma (the volume of a unit of single-donor plasmamay vary from 200 to 300 mL), extent of leukodepletion and the effect ofthis filtration on the extent of depletion of various coagulationfactors.

The filter device shown in FIG. 1 consists of a cylindrical housing 1and cover 3 fitted with inlet 2 and outlet 4 tubing connectors. Thehousing holds layers of filter media 35 mm in diameter. The top layer 6is a nonwoven fabric. Under this is a layer 8 of activatedcarbon/cellulose composite medium for methylene blue removal and up tofour layers 10 of a nonwoven filter medium, which can be made frompolypropylene, polyester, glass, and cellulose acetate components, forremoval of leukocytes and lipids, and for polishing filtration. In thefilter used in the examples, the carbon/cellulose composite was Carbac2640FH™, available from Cellulo Company Cranford, N.J.! and the nonwovenfilter for leukocytes was Type 825B from Lydall, Inc. Hamptonville,N.C.!. The base of the cylindrical housing 1 has a spiral filtratechannel 12 to improve air removal and draining efficiency.

The filter device is connected at its inlet to a sealed,sterile-dockable tubing about 40 cm in length, and optionally a tubingclamp. The filter is connected from its outlet to a receiving blood bagwith about 50 cm length of tubing. When the inlet tubing issterile-docked to a supply bag of virally inactivated plasma, thedistance measured from the midpoints of the supply and receiving bags isnominally 75 cm.

A unit of fresh-frozen plasma is thawed and brought to room temperature,and tare weighed. An aliquot of methylene blue solution is injected intothe plasma corresponding to a final dye concentration of 0.1 μM(equivalent to 0.4 μg/mL). The plasma is mixed by manual agitation ofthe bag for about 30 seconds, and a sample is removed for analysis. Thisplasma supply bag is sterile-docked to the filter assembly via theclamped filter inlet tubing. The supply bag is hung on a stand, with thefilter and supply bag suspended freely below. The tubing clamp is openedto start the flow of plasma. Total time for the entire unit of plasma topass through the filter is monitored. The filtered plasma is sampled foranalysis.

Examples of 1 to 10 utilized devices containing one layer of activatedcarbon filter and four layers of the nonwoven leukocyte filter; example11 utilized two layers of leukocyte filter medium. Examples 1 to 4illustrate methylene blue removal; examples 5 to 8 illustrate methyleneblue removal and changes in coagulation factors before and afterfiltration; example 9 shows methylene blue removal and leukodepletion(equivalent to 99.94% removal); examples 10 and 11 show the effects ofdifferent filter configurations and plasma temperature variation onmethylene blue removal and leukodepletion. For these tests pooled plasmawas used immediately following thawing (temperature at start offiltration cycle was about 4° C.).

    ______________________________________                                                           Methylene blue                                                                concn. in filtered                                                  Plasma    plasma (μg/mL)                                          Example  wt. (g)    a!         Filtration time (min)                          ______________________________________                                        1        300       <0.02       17.5                                           2        300       <0.02       14.9                                           3        205       <0.02       10.4                                           4        212       <0.02       10.6                                           5        264       <0.02       13.0                                           6        189       <0.02       11.3                                           7        199       <0.02       8.7                                            8        211       <0.02       13.5                                           9        169       <0.02       9.6                                            10       242       <0.02       39.6                                           11       287       <0.02       50.7                                           ______________________________________                                    

    __________________________________________________________________________                            Leukodepletion                                                                performance                                           Change in coagulation factor content (%)                                                              WBC count                                                                            WBC count                                           Fibrino-                                                                          Factor         per mL before                                                                        per mL after                                   Example                                                                            gen V  VII                                                                              VIII                                                                             IX XI filtration                                                                           filtration  b!                                 __________________________________________________________________________    2                                                                             3                                                                             4                                                                             5    -3  2  5  -3 -15                                                                              -48                                                      6    0   -14                                                                              9  -6 -11                                                                              -54                                                      7    0   -3 8  -8 -11                                                                              -62                                                      8    -3  0  8  -6 -15                                                                              -53                                                      9                       87800  50                                             10                      7400   0                                              11                      7400   0                                              __________________________________________________________________________      a!: 0.02 μg/mL is the limit of detection of methylene blue by HPLC         b!: Nageotte method. (American Assoc. of Blood Banks Technical Manual; p     760; method 11.12)                                                       

We claim:
 1. A method for simultaneously removing leukocytes andmethylene blue from plasma comprising passing said plasma through afilter comprising (a) a material capable of retaining leukocytes and (b)activated carbon.